Managing gene flow: a prerequisite for recombinant DNA biotechnology

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Managing gene flow: A prerequisite for

recombinant DNA biotechnology

By

Lukeshni Chetty

Submitted in fulfilment of requirements for the degree Philosophiae Doctor

Faculty of Natural and Agricultural Sciences, Department of Genetics

University of the Free State

Promoter: Professor C.D. Viljoen

Bloemfontein

South Africa

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A humble offering, placed at the Lotus Feet of

Bhagavan Sri Sathya Sai Baba

and so,

it is with love and servitude that I dedicate this research to the many

subsistence farmers of Africa. You are the ancient foundation of our

continent and this research was performed with a fervent hope of

enlightenment and as a modest attempt to lighten some of your plight.

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ACKNOWLEDGEMENTS

I would like to extend my sincere gratitude to the following people and institutions for their support throughout this research. The completion of such research is not without the insurmountable support by individuals and institutions alike.

Pannar for seed, field trial area and plant breeding expertise and advice. Also for their willingness to help, plant and maintain the field trials. A special thanks to: Willem Boshoff, Andre du Toit, Anthony Jarvie and Casper Benecke.

Prof. Charl van Deventer for planting and taking care of the Waterbron trial. NRF for financial assistance in way of scholarships

CIB for financial assistance in way of scholarships and research funds. To all those who worked selflessly and tirelessly on my fields, for not

complaining on those hot African days - I thank you, Japie, Gerhard, Natalie, Erin and Prof. C.D. Viljoen.

To my friends, who have throughout my research and without fail, supported and encouraged me. For your love and kind words - I thank you, Yanna, Kulsum, Japie, Natalie, Gerhard, Barbara, Elizma, Liezel, Sadie, Anthia, Lindy-Joy, Danisha, Bhavini and last but not least Parvershree & Kiru.

Willem Boshoff (Medical physics) for the assistance with statistical analysis. My Promotor, Prof. C.D. Viljoen, for the opportunity to do this research and

for your support and tireless efforts to bring this research to completion. My parents and family for your unconditional love and support throughout

my life.

The Departments of Genetics (Faculty of Natural and Agricultural Sciences) and Haematology and Cell Biology (Health Faculty) at the University of the Free State.

My beloved Bhagavan Sri Sathya Sai Baba for the strength to perform this research with love and courage.

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ABBREVIATIONS AND ACRONYMS

Bt Bacillus thuriengensis

CTAB Cetryltrimethylammonium bromide DNA Deoxyribonucleic acid

E East

EDTA Ethylene diamine tetra acetic acid ENE East-north-east

ESE East-south-east g/l Grams per litre GM Genetically modified

GMO Genetically modified organism

Ha Hectares HT Herbicide tolerance i.e. id est IR Insect resistance k Thousand L Litre

LOD Limits of detection

M Molar

m Metre

m2 Metre square

mg Milligram

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min Minute

mM Millimolar

m/s Metres per second

N North

NaCl Sodium chloride

NE North-east

NNE North-north-east NNW North-north-west

NW North-west

PCR Polymerase Chain Reaction

pH Percentage hydrogen

RH Relative Humidity rpm Revolutions per minute

S South SE South-east sec Second SSE South-south-east SSW South-south west SW South-west V Volts W West WNW West-north-west WSW West-south-West

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TE Tris-EDTA

TRIS Tris (hydroymethyl) aminomethane

µg Micro-gram

µl Micro-litre

ºC Degree Celsius

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LIST OF FIGURES

Figure 2.1.1 The 2007 biotech crop production in all 23 countries including the area and crop planted (James, 2007) . . . . . 11

Figure 2.1.2 Diagrammatic representation of the impact of agricultural biotechnology in regulatory frameworks, agriculture, the economy, the environment

and society . . . . . . . . . 12

Figure 2.3.1 Diagram represents the various crop production systems and the

levels of segregation . . . . . . . 24

Figure 3.1 Schematic of the soybean field trials in Delmas and Greytown (2005/2006). The cardinal directions are indicted for each location . 61

Figure 3.2 Schematic of the soybean field trials in Delmas and Greytown (2006/2007). The cardinal directions are indicted for each location . 62

Figure 3.3 The Vantage Pro mobile weather station situated on the field during

the flowering period . . . . . . . . 63

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Figure 3.5 Wind rose indicating the wind frequency during the two flowering days in Delmas during the (2005/2006) and (2006/2007) seasons . . 64

Figure 3.6 Wind rose indicating the wind frequency during the two flowering days in Greytown during the (2005/2006) and (2006/2007) seasons . 64

Figure 3.7 Control GM seed (A) and non-GM seed (B) after treatment with

Glyphosate solution (3%) . . . . . . . 65

Figure 3.8 Genotype detection. Lane 1 and 2 (negative sample), Lane 3 and 4 (positive sample - 129 bp), Lane 5 and 6 (negative control) and Lane 7 and 8

(positive control) . . . . . . . . 65

.

Figure 4.1 Field trial schematic for Bainsvlei (2005/2006) and (2006/2007)

. . . . . . . . . . 81

Figure 4.2 Field trial schematic for Waterbron (2006/2007). The surrounding non-GM maize fields were planted, a minimum of 4 weeks prior to the study Trial

. . . . . . . . . . 82

Figure 4.3 Total amount of pollen per distance interval over five days during flowering for Bainsvlei (2005/2006) . . . . . 83

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Figure 4.4 Total amount of pollen per days for five days during flowering for

Bainsvlei (2005/2006) . . . . . . . 83

Figure 4.5 Total amount of pollen per distance interval over five days during flowering for Bainsvlei (2006/2007) . . . . . 84

Figure 4.6 Total amount of pollen per days for five days during flowering for

Bainsvlei (2006/2007) . . . . . . . 84

Figure 4.7 Total amount of pollen per distance interval over five days during flowering for Waterbron (2006/2007) . . . . . 85

Figure 4.8 Total amount of pollen per day for five days during flowering for

Waterbron (2006/2007) . . . . . . . 85

Figure 4.9 Wind roses for five days during flowering in Bainsvlei (2005/2006)

. . . . . . . . . . 86

Figure 4.10 Wind roses for five days during flowering in Bainsvlei (2006/2007)

. . . . . . . . . . 87

Figure 4.11 Wind roses for five days during flowering in Waterbron (2006/2007)

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Figure 5.1 Diagram represents the cardinal directions that sampling was performed in all the field trials . . . . . . 106

Figure 5.2 Average percentage out-crossing over distance for Bainsvlei

(2005/2006) . . . . . . . . . 107

Figure 5.3 Percentage out-crossing for 16 directions over distance in Bainsvlei (2005/2006) with the power trendline and equation . . . 108

Figure 5.4 Average percentage out-crossing over distance for Bainsvlei

(2006/2007) . . . . . . . . . 109

Figure 5.5 Percentage out-crossing for 16 directions over distance in Bainsvlei (2006/2007) with the power trendline and equation . . . 110

Figure 5.6 Percentage out-crossing over distance for Waterbron (2006/2007)

. . . . . . . . . . 111

Figure 5.7 Percentage out-crossing for 16 directions over distance in Waterbron (2006/2007) with the power trendline and equation . . . 112

Figure 5.8 Out-crossing (

_■

) observed in Bainsvlei (2005/2006), Bainsvlei (2006/2007) and Waterbron (2006/2007) with the corresponding

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Figure 5.9 Temperature for five flowering days in Bainsvlei (2005/2006)

. . . . . . . . . . 114

Figure 5.10 Relative humidity for five flowering days in Bainsvlei (2005/2006)

. . . . . . . . . . 114

Figure 5.11 Temperature for five flowering days in Bainsvlei (2006/2007)

. . . . . . . . . . 115

Figure 5.12 Relative humidity for five flowering days in Bainsvlei (2006/2007)

. . . . . . . . . . 115

Figure 5.13 Temperature for five flowering days in Waterbron (2006/2007)

. . . . . . . . . . 116

Figure 5.14 Relative humidity for five flowering days in Waterbron (2006/2007)

. . . . . . . . . . 116

Figure 5.15 Out-crossing observed during the duration of the study

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LIST OF TABLES

Table 2.4.1 Potential pollen-mediated gene flow research in maize 29

Table 2.4.2 Pollen-mediated gene flow research in maize . . 29

Table 2.4.3 Pollen-mediated gene flow research in soybean . 30

Table 3.1 Soybean field trial phenology for Delmas and Greytown in the (2005/2006) and (2006/2007) planting seasons . . . . 57

Table 3.2 Pollen counts from traps for Delmas and Greytown in the (2005/2006)

and (2006/2007) seasons . . . . . . . 58

Table 3.3 Phenotypic and genotypic analysis for soybean seeds harvested from non-GM fields in Delmas and Greytown during (2005/2006) and (2006/2007)

seasons . . . . . . . . . 59

Table 3.4 Average temperature and relative humidity in Delmas and Greytown

for two days in two seasons . . . . . . 60

Table 4.1 Maize field trial phenology for the 2005/2006 and 2006/2007 planting seasons in Bainsvlei, Kroonstand and Waterbron . . . 77

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Table 4.2 Distance intervals for the pollen traps at the two locations 78

Table 4.3 PCR results for 35S detection in trapped maize pollen for Bainsvlei

(2005.2006) and (2006/2007) . . . . . . 79

Table 4.4 PCR results for 35S detection in trapped maize pollen for Waterbron

(2006/2007) . . . . . . . . . 80

Table 5.1 Calculated theoretical distances for 1%, 0.1%, 0.001% and 0.0001% out-crossing for Bainsvlei (2005/2006) . . . . . 103

Table 5.2 Calculated theoretical distances for 1%, 0.1%, 0.001% and 0.0001% out-crossing for Bainsvlei (2006/2007) . . . . . 104

Table 5.3 Calculated theoretical distances for 1%, 0.1%, 0.001% and 0.0001% out-crossing for Waterbron (2006/2007) . . . . . 105

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PREFACE

Genetically modified organisms (GMOs), refers to organisms that contains a transgene which was developed using recombinant DNA technology. This technology has mostly been applied to food crops such as maize and soybean, conferring transgenes with beneficial traits so as to increase crop yield, reduce input costs as well as reduce impact on the environment. In view of the substantial impacts agriculture has on biodiversity, GMO crop seemed a panacea. However, since its introduction, genetically modified (GM) crops have been surrounded by much controversy, as the unforeseen impacts in terms of environmental risks, human health, socio-economics and intellectual property rights to just name a few have plagued these crops. A great deal of research into the GM crop risk factors is required so that the safe use of this technology can be implemented.

Gene flow in GM crops, specifically pollen-mediated gene flow has been recognised as a potential area of risk in terms of the environment and human health. Adventitious commingling of GM maize or soybean with non-GM varieties, land races or wild relatives could result in compromised niche markets, carry health risks (pharmaceutical or industrial traits) or negatively impact the environment. Thus it is important to understand the factors affecting maize gene flow to be able to manage any potential negative impacts thereof.

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Despite the commercial propagation of GM maize and soybean for 10 years in South Africa, which includes a high adoption rate, very little research and no published data is forthcoming on the potential impact of GM gene flow in maize and soybean in South Africa. In this thesis, I have endeavoured to provide basic data with regard to GM gene flow which in hindsight should have been used to inform regulatory decisions over the last 10 years, regarding the release and management of GMOs, in order to be able to manage the technology and minimise risks to the environment and human health.

The thesis contains a literature review, three research chapters and a concluding chapter in which I make specific recommendations on management practice to minimize gene flow where necessary. The Literature review contains four sub-sections that have been or are in the process of publication. In this chapter, all figures and tables are contained within the text to maintain an easy reading style. The research chapters are written in article format and the figures and tables have been placed after the reference list. When reading this thesis you will experience some repetition between the introductions in the different research chapters – the reason for this is to place each research question within the correct context. Furthermore, the soybean research on potential pollen-mediated gene flow and pollen-mediated gene flow has been combined into one chapter. However, I felt that the corresponding chapter for maize was too cumbersome, in terms of the volume of data, and have separated these aspects into distinct chapters.

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Any research on the impact of genetic engineering is going to be controversial, depending on your point of view. In this thesis, I have attempted to traverse the path less known and provide some very basic yet essential answers to some of the most obvious, yet overlooked questions that should be asked including:

• Does out-crossing occur in self-pollinating soybean?

The basis of this question is that most if not all of the soybean varieties grown in South Africa are considered self-pollinating and gene flow is not considered significant. However, there is no evidence for this.

• What are the factors affecting gene flow in maize and how much of an impact does it potentially have?

There is very little consideration in South Africa on the need to minimize gene flow in maize – if only for niche non-GM markets. Most farmers do not apply any management strategies to minimize cross pollination and seed producers generally use regimes to ensure 96% to 99% seed purity.

• What practical management practises could be applied to minimize GM commingling?

The tolerance level of commingling often depends on the specific GM or its use. For example, for a field trial of a GM crop producing a pharmaceutical, commingling should not be allowed. However, depending on GM labelling requirements for approved GM crops, low levels of commingling might be acceptable.

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When you read this thesis, please consider for a moment the importance of trying to answer the very basic yet most fundamental questions regarding the introduction of GMOs into our environment: What is the impact of this technology considering the simplest of biological process – gene flow?

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CHAPTER 1: GENERAL INTRODUCTION

In the 2008/2009 planting season, South Africa entered the 11th year of growing GM (genetically modified) crops (James, 2007). The continued increase in the adoption of biotech crops is an indication that GM crops have been well received in South Africa compared to the rest of the continent that chooses a more conservative approach (James, 2007).

South Africa has commercialized GM crops since 1997 and insect resistant (IR) maize and cotton, herbicide tolerant (HT) soybean as well as stacked traits (IR and HT) for maize and cotton have been approved for general release (James, 2007). Despite this, there are a number of concerns surrounding the introduction of GM crops that need to be addressed. The intention with GM crops is to have a positive impact in terms of production, food security and the environment compared to conventional agricultural practice that is widely acknowledged as damaging to the environment (Carvalho, 2006; Castle et al., 2006). However, GM technology has also introduced additional complexities that cannot be ignored:

• Intellectual property rights and royalties

• The impact of GM on non-GM crop production in terms of niche markets

• Environmental impacts of GM compared to conventional agricultural practice

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Coexistence of GM crops with its conventional counterpart is generally overlooked. Non-GM products have become a niche market due to the introduction of GM. Furthermore, the commingling of undesired second or third generations GMOs in the food or feed market would be unacceptable as it could have dire consequences on human and animal health as well as the environment (Marvier and Acker, 2005; Moschini, 2006; Spok, 2007).

One foremost aspect of coexistence is gene flow, in particular pollen-mediated gene flow (Jank et al., 2006; Moschini, 2006; Lee, 2008). This has been largely neglected, probably due to the lack of understanding its importance. The aim of this study was:

1) To combine molecular techniques with field trials to study the self-pollinating nature of soybean and determine the extent of maize pollen movement and out-crossing under South African environmental conditions.

2) To make recommendations based on the data generated, on how pollen-mediated GM gene flow to non GM varieties or landraces can be minimized where necessary.

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CHAPTER 2: LITERATURE REVIEW

2.1 The overall impact of recombinant DNA biotechnology in agriculture

Three decades have passed since the development of recombinant DNA technology and its impact on various areas of science and society is evident (Cohen et al., 1973). Recombinant DNA refers to a DNA construct that contains a fragment of DNA from a foreign source, which once incorporated into the genome of an organism, is known as a genetically modified organism (GMO). This breakthrough, a mere two decades after the discovery of the structure of DNA (Watson and Crick, 1953), has made ground-breaking advances in the medical and agricultural sciences. In agriculture, recombinant DNA technology has added a new dimension to crop improvement, giving rise to biotech crops.

Due to an expanding global population, the agricultural industry is under constant pressure to increase food production (Endo and Boutrif, 2002). Currently, the world population is approximately 6.5 billion and is predicted to soar to an approximate 8.9 billion by 2050 (UN Report, 2004). Furthermore, it is predicted that global warming will also adversely affect agricultural production especially in developing countries (Houghton, 2005, Mendelsohn et al., 2006; Schlenker et al., 2006). Since the implementation of recombinant DNA in agriculture, it has been strongly suggested that biotech crops will aid in the alleviation of hunger and poverty (Endo and Boutrif, 2002), by developing crops with increased yield and low input costs

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such as insect resistance and herbicide tolerance. Whether this highly publicised benefit of GM crops will hold true for the impoverished, has yet to be determined.

In 2007, GM crops accounted for 114.3 million hectares in 23 countries (12 developing and 11 industrial) compared to 221.8 million hectares conventional crops (Fig. 2.1.1), representing 34% of global agriculture. Since its introduction in 1996, the area planted of GM crops has increased nine-fold in the world (James, 1997). Currently, the major GM crops are canola, cotton, maize and soybean. In the 2007 production season in South Africa, GM crops made up 80% of soybean (herbicide tolerance), 90% of cotton (insect resistance and herbicide tolerance) and 57% of white and yellow maize (insect resistance and herbicide tolerance) (James, 2007). South Africa remains the only country in Africa to commercially produce GM crops and in 2007 contributed approximately 1.8% to the global production of biotech crops. South Africa has annually increased GM crop production since 1997 and the adoption of second and third generation GMOs is imminent.

First generation GM crops are those with agronomic traits, for example, insect resistance or herbicide tolerance. Second generation GM crop have value-added traits for consumers such as enhanced nutritional value and third generation GMOs are aimed at producing pharmaceuticals or compounds for industrial use (Smyth et

al., 2002). Second generation GMOs have the potential to provide consumers with

vitamin enriched food (Falk et al., 2002) while third generation GMOs provide the prospect of low-cost drugs (Twyman et al., 2003; Elbheri, 2005). The envisioned

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plant-made pharmaceutical for infectious diseases may soon be a reality that South Africa would be amiss to ignore (Elbheri, 2005).

Despite the proposed benefits of biotech crops including the alleviation of poverty and hunger, there are many considerations surrounding GMO adoption. These include the impact on society, the environment, the economy, and agriculture (Fig. 2.1.2). Furthermore, the increased adoption of GM as well as the development of second and third generation GMOs presents a number of concerns regarding safety and challenges for coexistence. These concerns include lack of consideration for regulatory frameworks, intellectual property, cost benefit, the requirement for identity preservation in the development of niche non-GM markets, societal issues including acceptance, ethics and socio-economics as well as protection of the environment.

• Regulatory frameworks: The purpose of a regulatory framework is to manage the development and introduction of GMOs into the environment and to be able to capitalize on the potential benefit of this technology, while curtailing possible risks to human health and the environment. Regulatory frameworks tend to be specific to the needs of each individual country and often differ in terms of approach and stringency. The Cartagena Protocol on Biosafety is an instrument to assist developing countries when introducing GM crop. It imposes minimum regulatory requirements that must be incorporated into the framework but leaves the application to each adoptive country.

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• Intellectual property rights: The patenting of novel gene sequences and the requirement to pay royalties raises a concern at the farm level, with regard to seed saving and sharing which is culturally significant, especially in developing countries.

• Cost benefit: One of the primary aims of developing GM crops was to reduce input costs by among others reducing pesticide usage. Current GMOs provide potential cost benefits to farmers, including the subsistence farmer, but not to the consumer. However, the impact of farming subsidies in developed countries compared to the lack thereof in developing countries is seldom taken into consideration during agricultural cost analysis. Furthermore, the reaction of the market to GM crops is also not considered.

• Identity preservation: The introduction of GM crops into existing agricultural practice has resulted in the need for a management system known as identity preservation (IP). The importance of IP is to maintain GM traits as well as ensure that conventional varieties remain non-GM in terms of market requirements. IP includes the farm level management of coexistence and segregation of which one of the most significant considerations is pollen-mediated gene flow.

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• Human health: At a societal level, many concerns have been raised, regarding the safety of recombinant DNA technology and the long term effect of human health are still largely unknown. Health concerns include: the potential allergenicity of GM food, transgene transfer from GM food to intestinal micro-flora, the occurrence of unintended effects as well as altered nutrition value (Kuiper et al., 2002). Although, Biotech companies perform risk assessments on the safety of GM food, the long term effects on human health are still unknown.

• Consumer acceptance: Current GM crops do not provide any benefit to consumers, except for the promise of cheaper food. Consumer rights are well established in most countries including South Africa and in many countries GM products are labelled in the same way as additives and colourants. In contrast to consumers in Europe, consumers in South Africa are largely unaware of the existence of GM, let alone the presence of GM products in the food chain (Rowland, 2002; Viljoen et al., 2006). Consumers determine what drives the market and consumer attitudes to GM food will prove the final determinant in the GM debate.

• Ethics: GM crops are often marketed on their potential to alleviate starvation in the developing world with specific reference to Africa. The marketing promises food security and cheaper food (Cohen, 2005). However, these promises have not yet been realized. Furthermore, it is incorrect to make

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comparisons between developing and developed countries in terms of food security and the impact of technology thereon since farmers in Africa do not receive subsidies like their counterparts in developed countries in order to make “cheaper food” a reality. In addition to this, the patenting of genes resulting in a technology fee makes the GM technology unaffordable for the “starving “masses.

• Socio-economics: Agriculture forms such an important aspect of South Africa’s economy that it is important to consider the socio-economic impact of introducing GM crops on farmers and small scale farmers. GM crops have the potential to improve the economic status of farmers through increased production and lower input costs but it can also have negative impacts in terms of the requirement to acquire chemical inputs for traits such as herbicide tolerance (Cohen, 2005). In addition, the development of GM seed has created niche markets in commodity trading for non-GM and organic products. It is also envisaged that value added GM traits such as vitamin-enriched products may prove desirable to consumers. However, most farmers in South Africa are not aware of the impact that planting GM versus conventional crops may have on their ability to sell their produce and issues of market acceptance, safety and patents are not even considered.

• Environment: It is argued that GM crops can do no more harm in terms of the environment compared to conventional farming. However, as this

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technology is relatively new, it is important to ensure that the environment is protected and biodiversity conserved.

Non-target organisms: Very little is known on the impact of GM, especially those producing endotoxins, on non-target organisms including microbes, non-Lepidoptera species and small vertebrates. Target insects: The recent development of resistance in the target

organism in South Africa may have important environmental implications (Van Rensburg, 2007).

Weediness: The introduction of GM traits such as herbicide tolerance and the subsequent increased use of herbicides may contribute to the development of weediness in crops as well as other plants such as Johnson’s grass (Clements et al., 2004).

Gene flow: Pollen-mediated gene flow impacts more than just the diversity of genes in landraces and/or wild relatives. GM gene flow to conventional non-GM or organic crops has important economic consequences due to the loss of market value for such products (Zepeda, 2006; Demont and Devos, 2008; Lee, 2008). A further important but little considered impact of gene flow, is its contribution to the development of resistance in the target insect through potential exposure to sub-lethal doses of toxin as a result of low levels of GM in saved seed or maize refugia where out crossing has occurred (Chilcutt and Tabashnik, 2004). Furthermore, there is the possibility of transgene escape via horizontal gene flow into soil bacteria which could alter the genetic capabilities of beneficial soil bacterium. Thus GM biotechnology

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could have serious impacts on the environment and this should not be taken lightly.

When GM crops were developed and subsequently first commercialised, it was not envisioned that it would impact so many aspects of society. The primary aim of GM crops as put forward by companies and protagonists was to alleviate hunger and poverty (Chetty and Viljoen, 2007). When initially released, the social, environmental, economic and regulatory implications of GM crops were not considered. Nonetheless, the impact in these areas is undeniable, and has to be dealt with in a proactive manner. Although it is often argued that many of the potential impacts are similar or more severe for current conventional farming practice, it must be noted that the introduction of GM technology has added a complexity that from published literature does not appear to have been considered.

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Figure 2.1.1 GM crop production (2007) in all 23 countries including the area and crop planted (reproduced from James, 2007).

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SOCIETY

ENVIRONMENT

ECONOMIC

AGRICULTURE

REGULATIONS

Biod ivers ity Eco log_y S oc io-_ec on om ics Trad e Cos t-bene fit Safety La be llin_g E th ics Awarenes s Cons umer righ t’s Nut ritio nal valu e Cos t of foo d Coex isten ce Produ ction practic e Intellectu_al property_right’s Identity preservation

GMOs

SOCIETY

ENVIRONMENT

ECONOMIC

AGRICULTURE

REGULATIONS

Biod ivers ity Eco log_y S oc io-_ec on om ics Trad e Cos t-bene fit Safety La be llin_g E th ics Awarenes s Cons umer righ t’s Nut ritio nal valu e Cos t of foo d Coex isten ce Produ ction practic e Intellectu_al property_right’s Identity preservation

GMOs

Figure 2.1.2 Diagrammatic representation of the impact of agricultural biotechnology in regulatory frameworks, agriculture, the economy, the environment and society.

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2.2 GM biotechnology: friend and foe?

Chetty and Viljoen (2007). South African Journal of Science. Vol. 103. 269-270.

The opinion piece “Biotech’s defining moments” indicates a frustration shared by many scientists (Miller, 2007). This discontent stems from a perception that regulation of biotechnology in the name of biosafety is futile and biosafety research excessive (McHughen, 2006; Miller, 2007). At the same time advocates of biosafety, are too easily branded as anti-biotechnology, unscientific and unnecessarily short-sighted. A number of important but contentious issues are currently being debated. These include:

1) A perception that Non-Government Organisations (NGOs) stigmatize genetic modification (GM).

2) Risk assessments do not make a positive contribution.

3) Distinguishing between GM and non-GM has no scientific basis. 4) Coexistence studies between GM and non-GM are unnecessary. 5) Some regulatory systems are scientific and others not.

6) The Convention on Biological Diversity (CBD) impedes genetic engineering research as well as its promotion in developing countries

7) Mandatory labelling is unscientific (Miller, 2007).

As a result, the GM biotech community appears to be at loggerheads with itself and sadly the potential benefactors of this technology in developing countries are the losers. It is therefore necessary to depolarize the debate so that the attempts to serve the interests of Africa are realised and make GM biotechnology a “friend”.

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Proponents of GM biotechnology are of the opinion that NGOs continually stigmatize and undermine public confidence in recombinant DNA technology (Miller, 2007). Ironically, there are as many NGOs that unscrupulously campaign that biotechnology is a “silver bullet” to alleviate hunger in developing nations without any scientific basis. Some of the unsubstantiated statements, referring to recombinant DNA technology, include: “The biggest threats that hungry populations currently face are restrictive policies stemming from unwarranted public fears.” (Prakash and Conko, 2004), “a growing number of agricultural researchers, food experts and policymakers are pointing to plant biotechnology as a critical tool that can help increase food production and alleviate hunger without depleting natural resource.”(Council for Biotechnology Information, 2007) and “As Kenya faces yet another famine, food experts say that irrigation and adoption of genetically modified (GM) crops could be the way out of the perennial hunger problem.” (Opiyo, 2004).

Antagonists, equally, express negative sentiment towards GM biotechnology such as “Genetic engineering in its present form cannot form part of the solution; it is part of the problem.” (South African Freeze Alliance on Genetic Engineering, 2007), “African countries are being targeted by the GM industry and its lobbyists with unprecedented backing from the US government. Even food aid has been used to push GM into Africa.” (GM Watch, 2007)or “It is clear that GM crops offer no benefits and cannot feed the world.” (Ho, 2007). Thus, propaganda on both sides of the argument contributes to a skewed public perception of GM

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biotechnology, creates confusion, mistrust and cynicism amongst consumers and scientists alike.

Many scientists who develop GMOs (genetically modified organisms) believe that risk assessments are unnecessary and/or go beyond what is required to establish a lack of risk (Miller, 2007). Nonetheless, risk assessments remain vital to determining human safety. For example, a transgenic soybean engineered to contain a protein from Brazil nut would have been fatal for those with nut allergies, had the necessary allergy studies not been performed during the risk assessment (Nordlee et al., 1996). However, there is a case where a risk assessment may have proved vital. In 1989, the Eosinophalia-Mayalgia Syndrome (EMS) epidemic in the US, caused by the GM dietary supplement L-tryptophan, resulted in 37 mortalities (FDA, 2001). It is not certain whether the risk assessment performed was insufficient or whether it was performed at all. Nevertheless, by suggesting that risk assessments are excessive, GMO advocates unwittingly impede biotechnology progress by implying that the technology is above risk or that they fear scrutiny. In addition to determining health safety, environmental risk assessment is just as important. The conservation of biodiversity, including the preservation of landraces is a global concern. A recent study in the US found an unreleased transgenic herbicide-resistant creeping bentgrass introgressed into wild populations (Reichman et al., 2006). Clearly, risk assessments are imperative and not futile if performed with diligence.

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There is a continued debate amongst scientists, as to whether a GMO is substantially equivalent to its non-GM counter-part. Substantial equivalence implies that a GMO, with the exception of the transgene, is not significantly different to its conventional counterpart. However, the application of Intellectual Property Rights (IPR) makes a clear distinction between GM and non-GM in terms of plant breeder’s rights and patenting. In fact, GM and non-GM are biologically dissimilar (one has a transgene) and the GM variety is subject to patent rights and technology fees. Thus, GM and non-GM are seen as different on more than just a biological level. Whether the scientific community agrees or not, the legalities of transgene technology prohibit classification of GM and non-GM as substantially equivalent.

The numerous examples of “gene escape” over the last few years indicate that coexistence of GM and non-GM crop requires careful management. In Nebraska 2002, Prodigene’s pharmaceutical maize commingled with soybean and in the same year in Iowa, cross-pollination with conventional maize occurred (Elbheri, 2005). Prodigene’s financial losses were in excess of US$ 3 million which included fines and clean-up costs. Similar incidents of accidental transgenic entry into the food chain have occurred with Starlink maize (CRS Report for Congress, 2001) and Liberty Link rice 601 (FDA, 2006). Clearly, there is an urgent need for management to allow for coexistence and minimise commingling. The entry of a pharmaceutical crop into the human food chain would have devastating implications in Africa, where the resources to deal with such a situation do not

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exist. Thus, the continued examples of gene escape suggest that more research is required to prevent transgene escape.

A sector of the biotechnology community believes that GMOs are unscientifically over-regulated while others feel that regulations are insufficient. The FDA procedure to regulate GMOs is not that of approval but rather a consultation process, which is voluntary. This involves an audit of a risk assessment based on information provided by the biotech company. “During the consultation process, the FDA does not conduct a comprehensive scientific review of data generated by the developer” (FDA, 1997). Whereas the European Commission requires verification of information provided and may additionally perform necessary food safety and environmental risk assessments before granting approval of a GMO (Official Journal of the European Union, 2003). In South Africa, the Department of Agriculture through the GMO Act 15 of 1997, also performs a risk assessment audit using independent scientific expertise (Department of Agriculture, 1997; Department of Agriculture, 2005). While some regulatory systems are more stringent than others, it is uncertain which of these is more scientific. In reality, bureaucratic requirements are no indication of scientific content.

The CBD and specifically the Biosafety Protocol are often seen as an attempt to hinder the spread and acceptability of biotechnology in developing nations (Miller, 2007). In reality, the Biosafety Protocol is a facilitation mechanism to help countries deal with the introduction of GM, through the implementation of GM regulatory frameworks (Convention on Biological Diversity, 2000). Thus, it would

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seem short-sighted of biotech companies, NGOs and scientists to view the Biosafety Protocol in a jaded light when the CBD has proven to be an effective enabling mechanism in developing countries.

Mandatory labelling of GMO products is criticised as unscientific and an unnecessary expense (Miller, 2007). Food products are already being labelled with regard to potential allergens, ingredients and nutritional value. In addition, market directed labels including Kosher, Halaal, vegetarian, fat-free, low-fat, cholesterol-free and gluten-free are globally accepted. Thus labelling food products with regard to GM content is no less scientific than other current market directed labels. Additional information regarding the GM status of a product would allow for consumer choice and possibly contribute to an awareness of GM (Viljoen

et al., 2006). However, to deny consumers the right of choice, between GM and

non-GM, in product selection is unreasonable and will taint biotechnology in the eyes of economically influential consumers.

In conclusion, biotechnology can potentially benefit developing countries, but within reason. To claim that starving millions will be saved and then charge a technology fee is paradoxical. In order for this technology to be beneficial, it is important that interested parties including NGOs, government organisations and scientists work proactively to resolve conflicts. In order to depolarise the current debate and fulfil the mandate of hunger alleviation in Africa a level of transparency and forthrightness from proponents as well as opponents of recombinant DNA

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technology is required. This would inspire public confidence and perhaps make biotechnology more palatable to Africa.

2.3 Ten years of GM crops - can we coexist?

In 2007, South Africa was positioned eighth out of 23 countries producing genetically modified (GM) crops (James, 2007). GM crop was introduced in 1997 and a decade later South Africa now produces insect resistant and herbicide tolerant cotton and maize, as well as herbicide tolerant soybean contributing 1.8% to global GM crop production (James, 2007). Despite South Africa’s positive adoption of GM and a decade of production, there is currently no emphasis on establishing management practices for effective segregation of GM and non-GM crop. Nonetheless, with the development of second and especially third generation GM crops, establishing systems for coexistence will become a necessity (Moschini, 2006).

Coexistence refers to the effective segregation of a specific GM trait from conventional and organic production. Furthermore, segregation from other GM traits in order to meet market requirements would allow farmers a production choice which in turn allows for consumer choice. Therefore, coexistence is about satisfying the rights of both producers and consumers in terms of niche markets (Brookes, 2004; Jank et al., 2006).

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Since before the introduction of GM, seed producers were, and still are, required to maintain seed purity levels. Seed purity levels typically range from 96 to 99% with an accepted varietal difference of 1 to 4% (Karrfalt, 2004; Zhou et al., 2006). However, after the introduction of GM, the definition for “varietal difference” has now also been expanded to reflect the adventitious presence of GM. However, due to trade regulations, requirements for organic and non-GM production and GM labelling, the tolerance levels for GM in non-GM seed is usually set at a lower threshold and can even be zero depending on the nature of the genetic modification (Demont and Devos, 2008). Non-GM or GM purity levels have to be strictly adhered to as any infringement could result in serious economic loss to the seed producer and/or farmer. The development of pharmaceutical and industrial crop GMOs has added an additional complexity to seed production and coexistence that may require zero tolerance in terms of adventitious GM to ensure human and environmental safety.

GM crop segregation is required as a result of the different types of GM crop approval (including trial release), the requirements of consumers and the use of GM crop for food or feed, respectively. This is due to the development of niche markets to maintain trait segregation, especially in the case of GM pharmaceuticals, industrial compounds and biofuels (Figure 1.3.1). Thus, there are various levels of segregation for organic and conventional crops, as well as first, second and third generation crops.

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Prior to the green revolution, “organic” production was applied but not characterized as such. While initially requiring the absence of typical inputs used in the green revolution, organic crop production now also includes a requirement for the absence of GM. Organic crop production in the European Union (EU) currently stipulates 0% GM (Demont and Devos, 2008). From 2009, regulations in the EU will allow the adventitious presence of up to 0.9% GM in line with the threshold level for GM labelling (Demont and Devos, 2008). In the United States, the accepted level of GM commingling for organic production is 5% according to USDA guidelines (United States Department of Agriculture, 2002). Currently in South Africa there is draft legislation for organic production that allows 0% of adventitious GM. Thus, due to these requirements, segregation systems have to be established and require some form of certification or verification to ensure compliance.

Ironically, GM crop production is having a similar impact on conventional production similar to what the green revolution did to establish organic. GM production has established non-GM conventional production as a niche market. The adventitious presence of GM in a conventional non-GM system could either occur due to contaminated seed, unintentional farm-level commingling or post-harvest mixing (Demont and Devos, 2006). In the EU, a crop may be considered non-GM if it contains less than 0.9% GM. Currently in South Africa there is no prescription regarding the adventitious commingling of GM crop. However, the Department of Agriculture applies a 1.0% threshold for the non-GM status certification of agricultural exports.

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First generation GM crops with input traits (insect resistant and herbicide tolerant) are regulated in terms of their application for either for food or feed. In the case of first generation GM crops, identity preservation is at the level of the GM event. Thus GM crops regulated for human consumption may enter the feed market without contravening regulation. However, GM events regulated for feed may not enter the food market and require segregation (Fig. 1.3.1).

Similarly, second generation GM crops which have been developed with value-added traits (vitamin-enriched) in food and feed are also regulated per GM event. Second generation GM feed crops will probably not be permitted to enter the human food chain. However, a value-added trait specifically engineered for human consumption may not have the same benefit for animals and it is likely that this type of GM event may also require segregation from animal feed unless it is shown that they are safe for animal consumption. Although as yet no second generation GM crops have been approved for commercial release, these will require segregation to maintain the value-added trait as well as ensure that it does not commingle with other food or feed.

The use of third generation GMOs to produce pharmaceutical and industrial compounds as well as for biofuels, is the natural progression of GM technology, but adds significant complexity to GM segregation practice. The slightest possibility of this type of crop commingling with food destined for human or animal consumption would be considered unacceptable. The safety implications and economic

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consequences could be disastrous. Therefore, strict segregation of third generation GMOs from all other crop production systems should be mandatory.

Compared to conventional agricultural systems, there is less tolerance for the environmental impacts of GM. The prospect of transgene transfer, to landraces and wild relatives is a great concern. The conservation of biodiversity is a global issue and GM crops can compromise the genetic integrity of wild relatives or landraces via gene flow. Although gene flow from GM crops to wild relatives or landraces is just as much a reality with conventional crops, the latter are not under the control of patents and the genes involved have originated from wild relatives. Unfortunately, gene flow has already been observed with maize landraces in Mexico and Bentgrass in the United States (Quist and Chapela, 2001; Reichman et

al., 2006). In Africa, indigenous crops such as sorghum and cassava are an

important genetic resource and must be protected from transgene introgression. Although not indigenous to Africa, landraces of maize have acquired cultural importance and are an important aspect of agro-biodiversity – especially among rural farmers. Thus, just as maize germplasm must be preserved in Mexico, the centre of origin for maize, maize landraces require preservation in Africa and it is important to establish the necessary measures to achieve coexistence.

Coexistence can best be achieved through segregation which can be implemented at various levels during crop production including cultivation, harvest and post-harvest (storage, transport and processing) (Jank et al., 2006). For example, volunteer GM plants can result in commingling via gene flow through

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cross-Non-GM ORGANIC CONVENTIONAL FOOD FOOD FEED FEED INDU STRI AL B_IO FU EL PHARMA 1st 2nd 3rd GM

X



X

ENVIRONMENT Non-GM ORGANIC CONVENTIONAL FOOD FOOD FEED FEED INDU STRI AL B_IO FU EL PHARMA 1st 2nd 3rd GM

X



X

ENVIRONMENT

pollination or seed during harvest. Pollen-mediated gene flow is one of the major contributing factors that compromise coexistence. Unfortunately the effect of pollen-mediated gene flow is often underestimated due to a lack of understanding as a result of a lack of research. Therefore, in order to implement coexistence measures at the most basic level i.e. farm-level, a proper understanding of pollen-mediated gene flow is required to answer the question: is it possible for GM and non-GM or organic crops to coexist?

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2.4 GM gene flow: Much ado about nothing?

Genetically modified (GM) crops are currently produced in 23 countries and GM production contributed 34% of global agriculture in 2007. Currently, insect resistance and herbicide tolerance make up 72.2% and 20.3%, respectively of traits used (James, 2007). Although not yet produced at a commercial level, food crops have been genetically engineered for nutritional enhancement as well as for industrial and pharmaceutical traits (Moschini, 2006). This together with the rapid increase of GM crop production in many countries including South Africa and subsequent impact on trade with countries exhibiting a preference for non-GM has heightened the awareness of commingling between GM varieties and conventional varieties. The key contributor to commingling is gene flow which occurs at the farm level during crop production.

Gene flow is the movement of genes from one population to another. Vertical gene flow, with specific regard to GM crop is achieved via pollen. GM gene flow can occur through pollen from volunteer GM plants or from an adjacent GM variety with synchronous flowering (Huffman, 2004). Thus, pollen-mediated gene flow (PMGF) plays a key role in the management of coexistence between GM and non-GM crops.

In nature, PMGF is essential to maintain genetic variation and diversity. In crop improvement, plant breeders utilise PMGF to develop commercially viable varieties. After a variety has been established, PMGF has to be minimised to preserve the

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genetic integrity of the new variety and maintain seed purity. Gene flow from GM crops can also result in an infringement of intellectual property rights for seed producers and compromise the integrity of non-GM or organic niche markets that would result in economic loss in terms of market rejection of the product (Demont and Devos, 2006; Lee, 2008). In addition to this, GM crops with pharmaceuticals and industrial compounds have to be managed and contained to ensure that the human and environmental safety is not compromised. Gene flow from a pharmaceutical GM crop, to a food crop could result in a major health risk as well as economic losses (Elbheri, 2005; Moschini, 2008).

There are various factors that influence pollen-mediated gene flow. The pollination mechanism relies on several vectors including wind, insects, birds and animals. Furthermore, the synchronous maturation of stigma and anther is required, as well as ample pollen production, that is viable and is dependent on environmental conditions (temperature and relative humidity) (Kerhoas et al., 1987; Schoper et al., 1987a; Schoper et al., 1987b; Roy et al., 1995; Aylor, 2004). In addition, for successful gene flow to occur, viable pollen must interact with a receptive stigma resulting in successful pollination and fertilization (Bhatia and Mitra, 2003). Thus, the diverse criteria required for out-crossing to occur makes studying PMGF extremely challenging.

The different criteria influencing PMGF has led to the utilisation of a diverse array of research methods. Potential pollen-mediated gene flow (PPMGF) is studied by

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modelling, mathematical simulation and pollen capture (Table 2.4.1) (Raynor et al., 1972; Kerhoas et al., 1987; Schoper et al., 1987a; Schoper et al., 1987b; Roy et al., 1995; Fonesca et al., 2002; Jarosz et al., 2003; Aylor, 2004; Fricke et al., 2004; Arrit et al., 2007). Research into PMGF involves measuring the extent of out-crossing over distance (Paterniani and Stort, 1974; Garcia et al., 1998; Burris, 2001; Jemison and Vayda, 2001; Luna et al., 2001; Aylor et al., 2003; Byrne and Fromhertz, 2003; Henry et al., 2003; Ma et al., 2004; Stevens et al., 2004; Porta et

al., 2008; Bannert and Stamp, 2007). In addition, computer modelling has been

used to predict theoretical distances at which PMGF can occur under different permutations of environmental conditions (Fricke et al., 2004). The purpose of these studies is to determine the factors affecting PMGF and establish isolation distances to minimise gene flow to within threshold levels (Lee, 2008; Demont and Devos, 2008).

Despite GM crops being produced in 23 countries, research into pollen-mediated gene-flow especially in maize and soybean (popular GM food crops according to production values) has been lacking (James, 2007). According to published data for maize, the furthest that out-crossing has been detected is 650 m (Henry et al., 2003). However, a range of different distances has been recorded depending on the field trial design and the environmental conditions (Table 2.4.2). Similarly for soybean, generally considered to be a self-pollinating crop, very few published studies have determined the effect of the environment on PMGF (Table 2.4.3). Nonetheless, Ray et al., (2003) found 0.3% out-crossing at 5.4 m and Abud et al., (2007) found out-crossing of 0.52% at 1 m in soybean. One possible reason for the

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lack of published data on out-crossing in genetically engineered crops, is that prior to the development of GM and hence a specific target sequence that could easily be identified, plant breeders relied mainly on morphological characteristics to determine seed purity. Thus, many of the recommendations to minimize gene flow such as isolation distances would have been based on less sensitive and robust non-molecular criteria.

After a decade of GM crops being commercialised in South Africa, there is still no published data (i.e. none which could be found after an extensive survey of the literature) regarding the extent of PMGF in either maize or soybean under South African conditions. Despite this laissez-faire (nonchalant) stance, the recent contamination of food crops with pharmaceutical GM maize in the US (Prodigene) (Elbheri, 2005) and the introgression of transgenes in Mexican landraces has most certainly created a sense of urgency for such research, especially in developing countries who have the most to loose in terms of niche markets (Quist and Chapela, 2001).

The introduction of biotech crops has most certainly added new complexities in a variety of areas that were not initially envisioned. The main areas of impact are in agriculture practice, regulatory frameworks, economic, environment and on society. Unfortunately, the polarized nature of the GM debate has distracted from scientific inquiry into these issues. With second and third generation GMOs on our doorstep, it is imperative to establish guidelines for coexistence

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Description of methodology Furtherest distance

moved Reference

Pollen dispersal and deposition 60 m Raynor et al. (1972) Effect of dehydration on pollen viability None Kerhoas et al . (1987)

Heat tolerance on pollen viability None Schoper et al. (1987a) Water and heat stress on pollen viability None Schoper et al . (1987b)

Effect of temperature on pollen viability None Roy et al . (1995) Pollen production and dispersal None Fonesca et al . (2002) Airborne concentration and deposition 30 m Jarosz et al . (2003) Atmospheric exposure on pollen viability None Aylor (2004)

Computer simulation of pollen dispersal 880 m Fricke et al. (2004) Numerical simulation of pollen dispersal None Arritt et al. (2007)

Description of methodology Furtherest distance

out-crossed Reference

Out-crossing with phenotype detection 34 m Paterniani and Stort (1974) Out-crossing with detassling (phenotype) 184 m Garcia et al . (1998)

Out-crossing with gentotypic detection 200 m Burris (2001) Out-crossing with phenotype detection 40 m Jemison and Vayda (2001) Out-crossing with phenotype detection 200 m Luna et al . (2001) Aerobiological framework to assess out-crossing None Aylor et al . (2003)

Out-crossing with phenotype detection 183 m Byrne and Freomherz (2003) Out-crossing with gentotypic detection 650 m Henry et al . (2003) Out-crossing with phenotype detection 48 m Ma et al . (2004) Out-crossing with detassling (phenotype) 300 m Stevens et al . (2004)

Out-crossing with phenotype detection 56.7 m Porta et al . (2008) Out-crossing with phenotype detection 371 m Bannert and Stamp (2007) Table 2.4.1 Potential pollen-mediated gene flow research in maize.

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Category Description of methodology Furtherest out-crossing distance

Percentage

out-crossing Reference

Insect-mediated Out-crossing with phenotype detection None 2.50% Ahrent and Cainess (1994) Insect-mediated Out-crossing detected with enzymatic assay None 9 -19% Fujita et al . (1997)

Insect-mediated Out-crossing detected with isozyme analysis None 0.73% Nakayama and Yamaguchi (2001) PMGF Out-crossing with phenotype detection 5.4 m 0.03% Ray et al . (2003)

PMGF Out-crossing with gentotypic detection 8 m 0.02% Abud et al . (2007)

* This study was performed using wild soybean (Glycine soja )

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2.5 REFERENCES

Abud, S., de Souza, P.I.M., Vianna, G.R., Leonardecz, E., Moreira, C.Y., Faleiro, F.G., Junior, J.N., Monteiro, P.M.F.O., Rech, E.L. and Aragao, F.J.L. 2007. Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil. Genetics and Molecular Research 6(2): 445-452.

Ahrent, D.K. and Caviness, C.E. 1994. Natural cross-pollination of twelve soybean cultivars in Arkansas. Crop Science. 34: 376-378.

Arritt, R.W., Clark, C.A., Goggi, A.S., Sanchez, H.L., Westgate, M.E. and Riese, J.M. 2007 Lagrangian numerical simulations of canopy air flow effects on maize pollen dispersal Field Crops Research 102: 151-162.

Aylor, D.E., Schultes, N.P. and Shields, E.J. 2003. An aerobiological framework for assessing cross-pollination in maize. Agricultural and Forest Meteorology 119: 111-129.

Aylor, D.E. 2004. Survival of maize (Zea mays) pollen exposed in the atmosphere. Agricultural and Forest Meteorology. 123: 125–133.

Bannert, M. and Stamp, P. 2007. Cross-pollination of maize at long distance. European Journal of Agronomy. 27: 44-51.

Managing gene flow: a prerequisite for recombinant DNA biotechnology